PROJECT SUMMARY/ABSTRACT Although recent findings have shown the importance of the newly discovered circular non-coding RNAs (circRNAs) in the process carcinogenesis, studies showing the expression of circRNAs by oncogenic viruses or the potential regulation of human circRNAs by this type of viruses have not been described. Our preliminary findings suggest the discovery of two viral circRNAs expressed by the high-risk Human Papillomavirus type 16 (HPV-16) and type 18 (HPV-18) in cervical cancer (CaCx) and head and neck cancer (HNSCC) cell lines (the first example of circular RNAs produce by DNA viruses). These viral circRNAs (hpv-circK and hpv-circD) are localized in the nucleus of HPV-positive cells suggesting a role in transcription regulation. Furthermore, knockdown of hpv16-circK reduces cellular proliferation in HPV-positive cells and overexpression of this viral circRNA increases proliferation. Additionally, we found that expression of HPV-16 oncoprotein E6 is able to change the global expression of human circRNAs in primary human cells. Some of these circRNAs such as circGSK3B, circMYBL2 and circFN1 are expressed from alternative splicing regions of genes involved in cell metabolism, cell cycle and tumor microenvironment regulation, respectively. Our overall goal in this project is to analyze the newly discovered HPV-encoded circRNAs and to understand the regulation of human circRNAs by HPV in cancer. Finding the molecular mechanism(s) used by these circRNAs will identify new therapeutic targets in HPV-related cancers and in other types of malignancies. In order to identify and understand the importance of these viral and human circRNAs, we will utilize robust experimental models and clinical samples by Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR), RNA Fluorescence In Situ Hybridization (RNA FISH), Chromatin isolation by RNA Purification (ChIRP), and siRNA knockdown and exogenous delivery of circRNAs. The overall results will link high-risk HPV infection with the expression of viral or host circRNAs, contributing to a framework for the identification of new therapeutic strategies with potential relevance to both HPV-positive and HPV-negative cancers. This project is innovative not only because it is the first study describing and characterizing the expression of viral circRNAs but also because previous research has largely focused upon the interactions between HPV and other types of non-coding RNAs, specifically microRNAs and long non-coding RNAs.